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Genetic toxicity: in vitro

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in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference Type:
study report

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
Quality statement has been provided
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrahydrogen [29H,31H-phthalocyanine-2,9,16,23-tetrasulphonato(6-)-N29,N30,N31,N32]cobaltate(4-)
EC Number:
EC Name:
Tetrahydrogen [29H,31H-phthalocyanine-2,9,16,23-tetrasulphonato(6-)-N29,N30,N31,N32]cobaltate(4-)
Cas Number:
Molecular formula:
tetrahydrogen [29H,31H-phthalocyanine-2,9,16,23-tetrasulphonato(6-)-N29,N30,N31,N32]cobaltate(4-)
Specific details on test material used for the study:
The test material was a black powder and in the solvent it formed a dark-blue solution.


Target gene:
Histidine operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 1537
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
Test concentrations with justification for top dose:
Doses used : 1.0, 10, 100, 500, 1000, 2500, 5000 and 10 000 µg per plate
A preliminary toxicity study conducted on the test material at 14 doses of 1.22 µg to 10 000 µg per plate using the strain TA-100, exhibited approximately 98% toxicity at 10 000 µg dose. As such, the mutagenicity assays were conducted at 8 doses of 1 µg to 10 000 µg per plate.
Vehicle / solvent:
Vehicle used corresponds to the solvent used for the test material (sterile distilled water)
Untreated negative controls:
Negative control is the solvent control
Negative solvent / vehicle controls:
The solvent control is the negative control
Positive controls:
Positive control substance:
other: 2-anthramine (ANTH)
Details on test system and experimental conditions:
All indicator strains are kept at 4 °C on minimal medium plates supplemented with a trace of biotin and an excess of histidine.
The assay was conducted using two plates per dose level, with and without metabolic activation.
The plates with plasmid carrying strains contain in addition ampicillin (25µg/ml) to ensure stable maintenance of plasmid pKM101. New stock culture plates are made as often as necessary from frozen master cultures or from single colony reisolates that were checked for their genotypic characteristics (his, rfa, uvrB, bio) and for the presence of plasmid. For each experiment, an inoculum from the stock culture plates is grown overnight at 37°C in nutrient broth.
The bacterial strains were cultured in Oxoid Media #2 (nutrient Broth). The selective medium was Vogel Bonner Medium E with 2% glucose. The overlay agar consisted of 0.6% purified agar with 0.5 mM histidine, 0.05 mM biotin and 0.1M NaCl.
The activation assay is run concurrently with the nonactivation assay, the only difference is the addition of 0.5ml of S9 mix.
Evaluation criteria:
Because the test material and the cells are incubated in the overlay for approximately 2 days and a few cell divisions occur during the incubation perdiod, the test is semiquantitative in nature. Altough, these features of the assay reduce the quantification of result, they provide certain advantages not contained in a quantitative suspension test : the small number of cell divisions permits potential mutagens to act in replicating DNA (often more sensitive than nonreplicating DNA), combined incubation of the test material and the cells in the overlay permits constant exposure.

Because the procedures are semiquantitative, most data sets are evaluated using the following criteria :
- Strains TA-1535, TA-1537, and TA-1538 : if the solvent control value is within the normal range, a test material producing a positive respond equal to three times the solvent control value is considered mutagenic
- Strains TA-38, TA-100 : if the solvent control value is within the normal range, a test material producing a positive response equal to twice the solvent control value for TA-98 and TA-100 is considered mutagenic.
- Pattern : because TA-1535 and TA-100 are both derived from the same parental strain (G-46) and because TA-1538 and TA-98 are both derived from the same parental strain (D3052), to some extent there is a built-in redudancy in the microbial assay. In general, the two strains of a set respond to the same mutagen and such a pattern is sought. Generally, if a strain responds to a mutagen in nonactivation tests, it will do so in activation tests. Occassionally, exception to this pattern may also be seen.

Results and discussion

Test results
Key result
Species / strain:
other: All strains
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
No increase of mutagenic activity is observed at any doses in all strains.
Refer to "picture 1" in illustration section below for results summary.

Applicant's summary and conclusion

The test material Cobalt Phthalocyanine Sulfonate did not exhibit genetic activity in any of the assays conducted in this evaluation, and was not considered mutagenic under these test conditions according to our evaluation criteria.

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